Hi,

Label your probe w/DIG
Deparaffinezed your tissue sections (from xyline or histosol to distilled
water) then equilibrate your tissue section w/tris buffer.
For optimum probe penetration to target tissues, sections on slides should
be permeabilized w/50ug/ml proteinase K in tris buffer for 45 min at 37 C.
After incubation with enzyme solution, rinse the tissues w/3 changes of PBS
(phosphate buffered saline) for 10 min prior to prehybridization.
Prehybridize your sections for 2 h to reduce background
Hybridize your sections, then stringency wash.

If you need further information, feel free to ask me.

Ilhan Altinok

-----Original Message-----
From: kkdulany@unmc.edu [mailto:kkdulany@unmc.edu]
Sent: Monday, October 04, 1999 3:17 PM
To: Dana Settembre
Cc: Histonet
Subject: Re: Bobrowitz-InSitu


I see the in situ question has arisen again.  We have still not gotten
anywhere with this and our pathologist is trying to be patient.  He wants
us to do in situ on pancreas slides, paraffin and formalin fixed.  We are
using an oligo probe to insulin and tail it with DIG.

Does anyone have a tried and true method for doing this type of in situ??
Marianne and I don't feel qualified to do this without  molecular
experience but seems like we have to try to learn it.

Karen Dulany  HTL (ASCP)
Eppley  Institute for Cancer Research
Omaha, NE
402-559-5123